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Nickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1), and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and CD spectroscopies, potentiometry, isothermal titration calorimetry, HPLC and ESI-MS. We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2, bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies.
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Human Annexins A1, A2, and A8 as Potential Molecular Targets for
Ni(II) Ions
Nina E. Wezynfeld, Karolina Bossak, Wojciech Goch, Arkadiusz Bonna, Wojciech Bal,
and Tomasz Frączyk*
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
SSupporting Information
ABSTRACT: Nickel is harmful for humans, but molecular
mechanisms of its toxicity are far from being fully elucidated.
One of such mechanisms may be associated with the Ni(II)-
dependent peptide bond hydrolysis, which occurs before Ser/Thr
in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8,
proteins modulating the immune system, contain several such
sequences. To test if these proteins are potential molecular targets
for nickel toxicity we characterized the binding of Ni(II) ions and
hydrolysis of peptides Ac-KALTGHLEE-am (A11), Ac-
TKYSKHDMN-am (A12), and Ac-GVGTRHKAL-am (A13),
from annexin A1, Ac-KMSTVHEIL-am (A21) and Ac-SAL-
SGHLET-am (A22), from annexin A2, and Ac-VKSSSHFNP-am
(A81), from annexin A8, using UVvis and circular dichroism
(CD) spectroscopies, potentiometry, isothermal titration calorimetry, high-performance liquid chromatography (HPLC), and
electrospray ionization mass spectrometry (ESI-MS). We found that at physiological conditions (pH 7.4 and 37 °C) peptides
A12, A13, A81, and to some extent A22 bind Ni(II) ions suciently strongly in 4N complexes and are hydrolyzed at
suciently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in
the context of specic biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II)
binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization
of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel
toxicity and help design appropriate cellular studies.
Nickel is toxic for humans, causing allergy, cancers of the
respiratory system, and other serious health problems.
Despite this, nickel is a component of stainless steel and other
alloys, which are found in coins, mobile phones, accessories,
jewelry, and many implantable materials. These objects and
materials remain in contact with human tissues and release
Furthermore, signicant amounts of this metal are
delivered into lungs from standard as well as electronic
cigarettes and from airborne dust.
Nickel, as presumably
the most frequent contact allergen, is responsible for substantial
health problems, as more than 5% of the general population of
many developed countries (e.g., Germany and Denmark) is
sensitized to this metal.
The prevalence of nickel allergy is
especially high (17%) among women.
Although many
hypotheses have been proposed for the mechanisms of
development of metal-induced diseases, such as nickel allergy,
there is a high possibility that many relevant mechanisms are
yet to be unveiled. One of such possibility may be related to
hydrolysis of a peptide bond catalyzed by Ni(II) ions.
The Ni(II)-dependent hydrolysis of peptide bond occurs in
amino acid sequences Yaa-Ser/Thr-Xaa-His-Zaa (where Yaa
and Zaa stand for any amino acid residue and Xaa means any
amino acid residue except Pro). The hydrolyzed bond is located
between the Yaa and Ser/Thr residues. The binding of Ni(II)
occurs through the formation of a four nitrogen (4N) square
planar complex (by the imidazole and amide of histidine, and
two preceding amides), which causes the bending of the
peptide chain, and places the nucleophilic Ser/Thr hydroxyl
group in a proximity of the peptide bond preceding this residue.
As a result, the acyl shift takes place with the formation of an
intermediate ester product, which subsequently undergoes
spontaneous hydrolysis in aqueous solution.
Sequences susceptible to Ni(II)-dependent hydrolysis are
present in many proteins, but only some of them can undergo
hydrolysis under physiological conditions. We demonstrated
that the type of amino acid residue close to the potential
hydrolysis site has a signicant inuence on the hydrolysis rate.
This eect is especially relevant for those amino acids that
neighbor the histidine residue, where bulky and hydrophobic
residues are preferred for the fast reaction.
The formation
of the initial 4N Ni(II) complex is prerequisite for the
hydrolysis to occur; thus, the accessibility of the potential
Received: August 19, 2014
Published: October 7, 2014
© 2014 American Chemical Society 1996 |Chem. Res. Toxicol. 2014, 27, 19962009
cleavage site for Ni2+ ions is required. The protein sequences
harboring the cleavage sites should therefore be exposed on the
protein surface and suciently exible to adopt the square
planar structure of the complex.
However, local bends of the
protein main chain may predispose it to such conformation,
resulting in the hydrolysis reaction faster than expected on the
basis of sequence alone.
Annexins are proteins that bind phospholipid membranes in
a calcium-dependent manner. This binding is performed by
their conserved C-terminal domain. The N-terminal domain
interacts with other proteins, depending on the annexin type.
The specicity of these interactions has an impact on roles
played by dierent annexins.
Functions of annexin A1 include
(1) regulation of the innate and adaptive immune systems,
(2) participation in the plasma membrane repair system,
helicase activity,
and more. Annexin A2 participates in
immunological processes.
It is worth mentioning that the
expression of annexin A2 was found to be induced by nickel in
human HaCaT keratinocyte line.
Annexin A8 is a poorly
characterized member of the annexin family. It is known to
bind F-actin (similarly as annexin A1 and A2
) and is
associated with late endosomes.
To nd out whether human annexins A1, A2, and A8 are
potential targets for the toxicity of Ni(II) ions we synthesized
peptides that represent fragments of these proteins potentially
susceptible to Ni(II)-dependent hydrolysis. We also synthe-
sized a reference peptide Ac-GGASRHWKF-am, with an amino
acid sequence corresponding to that of the positive hydrolysis
control sequence established in our previous studies. Molecular
modeling, UVvis and circular dichroism (CD) spectroscopies,
potentiometry, isothermal titration calorimetry, high-perform-
ance liquid chromatography (HPLC), and electrospray
ionization mass spectrometry (ESI-MS) were used to character-
ize Ni(II) binding to these peptides and Ni(II)-dependent
peptide bond hydrolysis.
Materials. N-α-9-Fluorenylmethyloxycarbonyl (Fmoc) amino acids
were purchased from Novabiochem (Merck). Triuoroacetic acid
(TFA), piperidine, O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluro-
nium hexauorophosphate (HBTU), triisopropylsilane (TIS), and
N,N-diisopropylethylamine (DIEA) were purchased from Merck.
Acetic anhydride was purchased from Sigma-Aldrich. TentaGel S
RAM resin was obtained from Rapp Polymer GmbH.
Molecular Modeling. Crystallographic structures of annexins A1,
A2, and A8 (PDB codes: 1MCX, 2HYU, and 1W45, respectively) were
taken to simulations performed in Discovery Studio 4.0 Visualizer.
Ni(II) ion with a square planar geometry was incorporated to
respective potential sites. Conformations of complexes were obtained
by geometry optimization utilizing Dreiding-like force eld.
Peptide Synthesis. All peptides were synthesized in the solid
phase according to the Fmoc protocol
using an automatic peptide
synthesizer (Prelude, Protein Technology). The syntheses were
accomplished on a TentaGel S RAM resin (RAPP Polymere
GmbH), using HBTU as a coupling reagent, in the presence of
DIEA. Both acetylation of the N-terminus and cleavage were done
manually. The acetylation was carried out in 10% acetic anhydride in
DCM. The cleavage was done by the cleavage mixture composed of
95% TFA, 2.5% TIS, and 2.5% water. Crude peptides were isolated
from cleavage mixtures by precipitation by the addition of cold diethyl
ether. Following precipitation, peptides were dissolved in water and
lyophilized. Finally, they were puried by HPLC, and their identities
were checked by ESI-MS, as described before.
UVvis and Circular Dichroism Spectroscopies. The UVvis
spectra were recorded on the LAMBDA 950 UV/vis/NIR
spectrophotometer (PerkinElmer) over the spectral range 330850
nm. The CD spectra were recorded on the J-815 CD spectrometer
(JASCO) over the spectral range 300650 nm. For both methods,
path length was 1 cm, and samples containing 0.95 mM peptide and
0.9 mM Ni(NO3)2were titrated with small portions of concentrated
NaOH in the pH range 3.011.5, at 25 °C.
Potentiometry. Potentiometric titrations were performed on a
907 Titrando Automatic Titrator (Metrohm), using a Biotrode
combined glass electrode (Metrohm), calibrated daily by nitric acid
One hundred millimolar NaOH (carbon dioxide free) was
used as a titrant. Samples (1.5 mL) were prepared by dissolving
peptides in 4 mM HNO3/96 mM KNO3to obtain 0.81.5 mM
peptide concentrations. The Ni(II) complex formation was studied
using samples in which the molar ratios of peptide to Ni(II) were
1:0.9, 1:0.45, and 1:0.3. The pH range for all potentiometric titrations
was 2.711.6. All experiments were performed under argon at 25 °C.
Three titrations were included simultaneously into calculations for
protonation, and ve for Ni(II) complexation. The data were analyzed
using the SUPERQUAD and HYPERQUAD programs.
deviations provided by these software and reported here have
statistical nature and do not include potential systematic errors.
Isothermal Titration Calorimetry. Calorimetric titrations were
carried out on the Nano ITC Standard Volume calorimeter (TA
Instruments). The sample cell (950 μL) was lled with a peptide
solution, and the reference cell was lled with Milli-Q water. The
syringe (250 μL) was loaded with a Ni(II) solution. Milli-Q water was
degassed under vacuum for 15 min before sample preparation.
Furthermore, peptide and Ni(II) solutions were degassed for 5 min
before loading into the cell and the syringe. The peptide solutions
contained 2 mM peptide, 100 mM H3BO3, and 64 mM KNO3at pH
9.0. The ionic strength was 0.1 M. The Ni(II) solutions contained 20
mM Ni(NO3)2and 64 mM KNO3. Typically, 2 μL of the Ni(II)
solution was added to the peptide solution at 12001500 s intervals
using a stirring speed of 250 rpm. To prevent the metal-dependent
hydrolysis of peptide bond during the experiments, the measurements
were performed at 5 °C. The blank linear functions were calculated on
the basis of the last measurement points, where the observed heat ow
resulted almost exclusively from the dilution of Ni(II) solution. The
blank corrections were made by subtracting values of the blank linear
function from the raw data. The data were analyzed by the
NanoAnalyze Software (TA Instruments).
HPLC Measurements of Hydrolysis Rates. Samples containing
0.8 mM peptide, 1 mM Ni(NO3)2, and 20 mM HEPES at pH 8.2 or
7.4 were incubated at 25, 37, 45, or 60 °C. Twenty microliter aliquots
were periodically collected from the sample and acidied by addition
of 20 μL of 2% TFA to break down hydrolytic Ni(II) complexes in
order to stop the hydrolysis reaction. The products of hydrolysis were
separated by HPLC and their identity checked on ESI-MS.
Kinetics Analysis. To determine rate constants for the hydrolysis
reaction we used the iterative optimization algorithm based on the
LevenbergMarquardt method, which was adapted to minimize the
squared distance between the experimental data and theoretical curves
obtained from numerical solutions of the corresponding system of the
following equations (eqs 13):
=− + −
R kSt St M S
d()( () )
4N 1 0 0 (1)
=− + − −
R kSt St M S kIPt
d()( () ) ()
4N 1 0 0 2 (2)
They were used to calculate the rst (k1) and the second rate
constant (k2) on the basis of changes in time of substrate (S),
intermediate product (IP), and products (P) concentrations depend-
ing on the ratio of 4N hydrolytic complexes to the rest of substrate
species (R4N) at a given pH, and the initial concentration of the
substrate (S0) and metal ions (M0). All calculations were performed in
the Wolfram Mathematica 8 environment.
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620091997
The full description of the model used to describe the kinetics of the
metal dependent hydrolysis, including approximations that could be
used when the concentration of 4N complexes are in excess or in
deciency to the rest of the substrate species throughout the
experiment, is available in the Supporting Information.
Analysis of 3D Structures of Annexins A1, A2, and A8.
Structures of human annexins A1, A2, and A8 are available in
the RCSB PDB database. This allowed us to analyze geometries
of potential sites of Ni(II) binding and Ni(II)-dependent
peptide bond hydrolysis. However, for human annexin A1, the
crystallographic data are available only for Cαatoms of the
main chain of the protein (PDB code: 1AIN). The amino acid
sequence alignment of porcine and human annexin A1 shows
high similarity (89% of identical amino acid residues), with full
identity in the regions with potential sites of Ni(II) binding and
hydrolysis. Structural alignment of the human (PDB code:
1AIN) and porcine (PDB code: 1MCX) proteins conrms high
similarity. Therefore, we used the structure of porcine annexin
A1 (PDB code: 1MCX), which includes bound calcium ions.
For annexin A2 we chose the crystal structure of human protein
complexed with calcium ions and heparin fragments (PDB
code: 2HYU). These heparin fragments do not aect the
conformation of potential Ni(II) binding and hydrolysis sites
(as compared to the respective structure without heparin; PDB
code: 2HYW). For annexin A8 we chose the structure of the
human protein without calcium ions (PDB code: 1W45), as
these ions do not inuence the site of potential Ni(II) binding
and hydrolysis (as compared to the respective structure with
calcium ions; PDB code: 1W3W). Moreover, the 1W45
structure contains a longer fragment of the N-terminus of the
protein where the potential Ni(II) binding and hydrolysis site is
To test the possibility of formation of 4N square planar
Ni(II) complexes in the above-mentioned annexins we
simulated the Ni(II) binding with the use of geometry
optimization utilizing Dreiding-like force eld
in Discovery
Studio 4.0 Visualizer. The fragments of proteins containing sites
for Ni(II) binding and hydrolysis, together with corresponding
simulated complexes are shown in Figure 1. All these sites are
located in coils, assuring the exibility of the polypeptide chain.
Accordingly, all simulated complexes were acquired without
disturbing large parts of proteins, which means that the process
of Ni(II) binding would not need to overcome excessive energy
barriers. This analysis shows that the sites are accessible for
solutes such as metal ions and can adopt conformations
conforming to square planar complexes. These features can
promote the binding of Ni(II) ions to potential hydrolytic sites
in annexins.
In the amino acid sequence of human annexin A1 there are
three Yaa-Ser/Thr-Xaa-His-Zaa sites that may potentially bind
Ni(II) and undergo Ni(II)-dependent peptide bond hydrolysis.
They are 101Thr-Gly-His103,244Ser-Lys-His246, and 291Thr-Arg-
His293 (Figure 1AF). They are located on the convex side of
the molecule. This side binds Ca2+ ions and interacts with
phospholipids. The following Ca2+ binding residues are located
in the proximity of potential Ni(II) sites: Lys97, Leu100,
Glu105, Asp253, Leu256, Gly288, Met286, and Gly290.
Although it is not observed in simulated structures, this
proximity suggests that the Ni(II) binding can aect Ca2+
binding and vice versa. Interestingly, imidazole groups of His103
and His293 residues are 4 Å apart and interact via ππ
Human annexin A2 exists in two isoforms resulting from
alternative splicing. The second isoform diers from the
canonical one by the longer N-terminus (18 residues). Here, we
will use the numbering for the canonical sequence. It contains
two Ni(II) hydrolysis sites. The N-terminal 3Thr-Val-His5site
is not resolved in crystal structures, indicating that it is exible
or assumes multiple conformations. Indeed, NMR and CD
spectroscopic characterization of the N-terminal fragment of
annexin A2 showed that, although it adopts an α-helical
conformation in a membrane-mimetic environment, it has
random structure in aqueous solution.
The 92Ser-Gly-His94
site is located on the surface of the convex side of molecule
(Figure 1G,H). Adjacent amino acid residues Lys88, Leu91,
and Glu96 bind Ca2+ ions. Furthermore, His94 interacts with a
fragment of heparin. Thus, the Ni(II) binding is likely to aect
interactions of annexin A2 with Ca2+ ions and heparin.
The potential Ni(II) binding sequence in human annexin A8,
18Ser-Ser-His20 (Figure 1I,J) is located on the concave side of
the protein, opposite to the Ca2+ binding side. The fragment is
on the surface of the protein in a coiled, exible structure.
The above analysis shows that all Yaa-Ser/Thr-Xaa-His-Zaa
sites in human annexins A1, A2, and A8 are easily accessible for
Figure 1. Fragments of human annexins A1, A2, and A8 with potential
Ni(II) binding sites. Shown are native proteins (A, C, E, G, I) and with
Ni(II) ion bound (B, D, F, H, J) as modeled with the use of geometry
optimization utilizing Dreiding-like force eld
in Discovery Studio
4.0 Visualizer. Shown are fragments with 101TGH103 (A,B), 244SKH246
(C,D), and 291TRH293 (E,F) from annexin A1, 92SGH94 (G,H) from
annexin A2, and 18SSH20 (I,J) from annexin A8. Amino acid residues
expected to compose the Ni(II) complex are shown in a stick
representation. The sequences studied in this article are violet; Ca,
green; Ni2+, yellow; heparin, white sticks.
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620091998
Ni(II) ions. Therefore, we decided to characterize the Ni(II)
binding and susceptibility to Ni(II)-dependent peptide bond
hydrolysis of peptides with sequences taken from the annexins.
Sequences of these peptides in the protein context and labels
used for them in the text are presented in Table 1.
Ni(II) Complexation. We used UVvis and circular
dichroism spectroscopies, potentiometry, and isothermal
titration calorimetry to characterize the Ni(II) binding to
annexin-derived peptides. Table 2 presents cumulative
logarithmic protonation constants of all peptides calculated
on the basis of potentiometric titrations. All peptides contain a
single His residue, with pKvalues in the range of 6.1 to 6.8.
Most of the peptides also contain a Lys or a Tyr residue, with
pK9.310.6, and acidic residues, with pK3.64.8.
As potentiometry records only H+concentration changes
upon the metal ion binding, rather than metal specic
properties, we utilized UVvis results to select the correct
stoichiometric model of Ni(II) binding and to verify Ni(II)
complex formation constants calculated on the basis of
potentiometric titrations. The distributions of Ni(II) species
described by particular models were compared to the pH
dependence of absorbance at the maximum of dd bands of
low-spin complexes. We used root-mean-square deviation
(rmsd) as a parameter to measure dierences between UV
vis spectroscopic parameters and species distributions based on
these models. Ni(II) can be coordinated by imidazole ring of
histidine residue (1N complex), by the imidazole and two
amide nitrogens (3N complex), or by the imidazole and three
amide nitrogens (4N complex). Previous research showed that
2N complexes are not observed in hydrolytic peptides
containing Yaa-Ser/Thr-Xaa-His-Zaa sequences and that 3N
complexes are formed from 1N species through cooperative
deprotonation of two amide nitrogens.
We examined at least
two models for each peptide. In the rst model we assumed the
formation of 1N, 3N, and 4N complexes. In the second model
we disregarded 3N complexes. The simulated amounts of 3N
complexes, whenever considered, were always low compared to
corresponding 4N complexes, and never exceeded 25% of all
Ni(II) species. We were able to conrm the existence of 3N
complexes in UVvis spectra only for A13, A21, A22, and
A81 peptides. The comparisons of Ni(II) binding models
considered are presented in Figure S1 of the Supporting
Information. The veried Ni(II) binding constants are
presented in Table 3. In Figure 2 we present examples of
Ni(II) species distributions compared to the absorbance at the
maximum of respective dd bands. At pH above 4, Ni(II)
binds to peptides via a His imidazole ring nitrogen (1N
complex). Its presence could not be conrmed for A21,
probably as a result of a lower concentration of this peptide in
potentiometric experiments, due to its low solubility. In general,
1N complexes did not exceed 20% of all Ni(II) species, which
could also signicantly hinder the detection of this complex for
the A21 peptide. As expected, the UVvis spectra at pH of
the maximum of molar fraction for 1N complexes did not dier
signicantly from those at pH below 4, which conrms that 1N
complexes are high-spin (octahedral), similarly to the Ni(II)
aqua ion.
The good t of the absorbance at the dd band maximum
with the sum of 3N and 4N complexes helped us conclude that
3N complexes of A13 and A81 peptides contain low-spin
Ni(II). On the contrary, for A21 and A22 peptides, the
absorbance of Ni(II) complexes at the dd band maxima
corresponds to the sum of 4N complexes rather than the sum
of 3N and 4N complexes. Thus, Ni(II) in 3N complexes of
A21 and A22 is high-spin.
Low-spin 4N complexes are hydrolytically active species.
Their Ni(II) complex formation constants are highlighted in
bold in Table 3. It is worth noting that almost every studied
peptide can form more than one 4N complex, due to
deprotonations of Lys or Tyr residues. The only exception,
Table 1. Sequences of the Studied Peptides
protein label amino acid sequence of
peptide position of the histidine
residue in the protein
A1 A11 Ac-KALTGHLEE-am 103
A12 Ac-TKYSKHDMN-am 246
A13 Ac-GVGTRHKAL-am 293
A2 A21 Ac-KMSTVHEIL-am 5
A22 Ac-SALSGHLET-am 94
A8 A81 Ac-VKSSSHFNP-am 20
The numbering from protein sequences including the initiator
methionine is shown.
The positions are valid for isoform 1, chosen as
canonical sequence from the two produced by alternative splicing.
The positions are from isoform 2.
Table 2. Logarithmic Protonation Constants for Annexin
Peptides Determined at 25 °C and I= 0.1 M (KNO3)
peptide HL H2LH
A11 10.18(1) 16.97(1) 21.78(1) 25.68(1)
A12 10.61(1) 20.99(1) 30.34(1) 36.70(1) 40.31(1)
A13 10.05(1) 16.13(1)
A21 9.85(1) 16.19(1) 20.51(1)
A22 6.66(1) 10.98(1)
A81 10.19(1) 16.56(1)
Standard deviations on the least signicant digits, provided by
are given in parentheses.
Table 3. Logarithmic Ni(II) Complex Formation Constants for Annexin Peptides Determined at 25 °C and I= 0.1 M (KNO3)
peptide NiH3L NiH2L NiHL NiL NiH1L NiH2L NiH3L
A11 n.d.
n.d. 12.61(2) n.d. n.d. 11.50(1) 21.59(1)
A12 32.43(3) n.d. n.d. 9.53(1) 0.08(1) 10.67(1) 21.44(1)
A13 n.d. n.d. 12.53(2) n.d. 3.13(1) 11.21(1) 21.61(1)
A21 n.d. n.d. n.d. n.d. 4.31(3) 12.11(1) 21.80(1)
A22 n.d n.d. n.d. 2.90(1) n.d. 13.33(2) 21.24(1)
A81 n.d. n.d. 12.55(3) n.d. 2.86(1) 10.86(1) 21.10(1)
Standard deviations on the least signicant digits, provided by HYPERQUAD,
are given in parentheses.
n.d.; not detected. Values for 4N
complexes are bold.
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620091999
the A22 peptide contains neither Lys nor Tyr residue and
forms only one 4N complex in the studied pH range.
The low-spin 3N and 4N complexes are detectable not only
in UVvis but also in CD spectra in the region specic for dd
transitions (350600 nm). While UVvis spectra of Ni(II)
complexes are very similar to each other for all studied
peptides, we observed a variety of CD spectral patterns,
especially in terms of the number of extrema (Figure 3). In the
range of 300650 nm, we observed just one minimum and one
maximum for peptides A11 and A13. The CD spectra of
Ni(II) complexes of A21 and A81 peptides have an
additional maximum at about 570 nm. A22 has one maximum
at 456 nm but also two much smaller minima at 385 and 562
nm. For the A12 complex we noticed three extrema of
comparable intensities.
On the basis of potentiometric data, we used the least-
squares calculations to deconvolute UVvis and CD spectra for
each low-spin complex species. Table 4 presents their molar
absorption coecients. For the A12 peptide overlapping
deprotonations of two Lys and one Tyr residues yielded four
4N species, which could not be deconvoluted reliably. In the
course of preliminary calculations we detected that two
intermediate species, NiH1L and NiH2L, may have very
similar spectra and consequently merged them in the
calculations. Generally, the spectra of dierent Ni(II)
complexes for the same peptide have similar patterns (see
Figures S2 and S3 of the Supporting Information). The
exception is the NiL complex of A12forwhichwe
distinguished an additional minimum of a very low intensity
at short wavelengths. The wavelengths of the maximum
absorbance (λmax)ofdd bands for a given peptide dier by
less than 4 nm for A11, A12, and A21 peptides, where,
according to stoichiometry of complexes, only 4N species are
low-spin. For A13andA81, where potentiometric
calculations indicated that both 3N and 4N complexes should
have low-spin character, the shift was 29 and 24 nm,
respectively. According to the ligand eld theory, an additional
amide nitrogen involved in the Ni(II) binding in 4N complexes
should cause a larger dierence in energy between d orbitals
than three nitrogen ligands as in 3N complexes. In other words,
bands of 4N complexes should be shifted toward shorter
wavelengths relative to the 3N complexes, as we indeed
observed for A13 and A81. This conrms that A13 and
A81 peptides form low-spin 3N complexes.
Calorimetric titrations were carried out at pH 9.0 to ensure
the high percentage of 4N complexes (>98%) and at 5 °Cto
inhibit the hydrolysis reaction. Thermodynamic parameters of
the analyzed process are presented in Table 5. The
representative titration is shown in Figure 4. We could not
obtain satisfactory results for Ni(II) binding to A21 due to
insucient solubility of this peptide. Conditional Ni(II)
complex formation constant values for studied peptides are in
the range 1.16.8 ×104M1. To compare binding constants
obtained by ITC and potentiometry, we calculated conditional
Ni(II) binding constants at pH 9.0 on the basis of
potentiometric results. They are 2040 times higher than
those obtained from ITC. This discrepancy could be a result of
an interaction between Ni(II) and the buer. Although we did
not detect such interactions in control titrations of borate with
Ni(II) ions, there is a literature report by Tilak et al.
indicating that borate and nickel form a weak Ni(H2BO3)2
Dening the association constant of the Ni(II) borate (B)
complex (Kbuff)byeq4,
buff 2
one can easily obtain a formula converting the apparent binding
constant of the Ni(II) complexation to the studied peptide
obtained from ITC (Kapp) into the conditional binding constant
at pH 9 (Kc,eq5)as
=KK K B(1 [ ] )
capp buff 2 (5)
Figure 2. Species distributions of Ni(II) complexes of selected peptides: Ac-KALTGHLEE-am (A11; left), Ac-KMSTVHEIL-am (A21; in the
middle), and Ac-VKSSSHFNP-am (A81; right), at 25 °C, calculated for concentrations used in UVvis and CD titrations (0.95 mM peptides and
0.9 mM Ni(II)) using stability constants from Tables 2 and 3. The common scale left-side axes represent Ni(II) molar fractions. Ni(II) species are
color-marked as follows: Ni(II) aqua ion, black; 1N complex, red; 3N complex, blue; 4N complexes, green and orange. Pink dotted lines show the
sum of 4N complexes, while navy dashed lines show the sum of 3N and 4N complexes. The variable scale right-side axes provides values of
absorbance and ellipticity at the dd band maximum of low-spin complexes: UVvis absorbance, black circles; CD ellipticity, blue, pink, and violet
triangles. Absorbance and ellipticity values are omitted for clarity. Species distributions for other peptides are available in Figure S1 of the Supporting
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620092000
We followed the assumption of Tilak et al. that only H2BO3
ions bind Ni(II).
Taking that pKof H3BO3at 5 °C is 9.4,
the log Kbuff explaining the discrepancy between ITC and
potentiometric results should be in the range of 4.44.7. This is
in a good agreement with results of Tilak et al. (log K= 3.8
Therefore, we can safely use ITC results to compare
Ni(II) anities of the studied peptides. In agreement with
potentiometric results, A12 and A81 peptides have the
highest anity to Ni(II) ions, followed by A22 and A13
peptides. A11 has the lowest anity for Ni(II) ions, with the
value of binding constant more than six times lower than for
A12 peptide.
The observed enthalpy changes (ΔHobs) range between 27.6
and 47.7 kJ ×mol1(Table 5). They have positive values,
indicating that Ni(II) coordination is an endothermic reaction.
The highest ΔHwas noticed for A81, the lowest for A11.
The value of the heat absorbed after addition of Ni(II) ions to
peptide solution is a result of Ni(II) binding to peptides and
the sum of energy of all other processes occurring after
injection, including dilution and buer protonation. The heat of
dilution was subtracted. The buer protonation was the result
of proton release from peptides upon Ni(II) binding. At pH 9.0
almost all histidine residues are deprotonated spontaneously, so
Ni(II) binding only requires deprotonation of three amide
nitrogens. According to literature data,
the enthalpy of borate
protonation at 5 °C is about 18.6 kJ ×mol1. Assuming that
released protons react only with the buer, the contribution of
this process is about 55.8 kJ ×mol1. These considerations
yield estimated values of Ni(II) binding enthalpy changes
(ΔHNi(II) bind) ranging between 83.4 and 103.5 kJ ×mol1
(Table 5). We did not include the possible protonation of
lysine or tyrosine residues, as we calculated that it has a minor
buering eect in experimental conditions because of a much
higher concentration of buer (100 mM) from that of the
peptide (2 mM).
The observed entropy changes (ΔSobs) range between 176.4
J×mol1×K1for A11 and 260.2 J ×mol1×K1for A81
(Table 5). The correction of enthalpy change values by the
buer protonation eect led to a corresponding increase in the
entropy changes associated with Ni(II) binding (ΔSNi(II) bind)
by about 200 J ×mol1×K1. It suggests that Ni(II) binding is
the entropy-favored reaction under ITC experiment conditions.
It is mainly the result of water release from nickel aqua ions
after Ni(II) binding to peptide.
Hydrolysis of Peptides. All studied peptides undergo
Ni(II)-dependent peptide bond hydrolysis. Ni(II) ions
selectively cleave a peptide bond preceding serine or threonine
residue in Yaa-Ser/Thr-Xaa-His-Zaa sequences. Separation of
Figure 3. CD spectra of low-spin Ni(II) complexes of peptides: Ac-KALTGHLEE-am (A11), Ac-TKYSKHDMN-am (A12), Ac-GVGTRHKAL-
am (A13), Ac-KMSTVHEIL-am (A21), Ac-SALSGHLET-am (A22), and Ac-VKSSSHFNP-am (A81), at dierent pH values, coded with
rainbow colors from red (the lowest pH 3) to dark blue (the highest pH 11.5). The spectra of apopeptides, exhibiting no extrema in this spectral
range, are marked by dotted gray lines (mostly not visible as they have zero value throughout all the spectra). The titrations were carried out at 25
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620092001
reactants by HPLC allowed us to detect four species in all
cases: a substrate, an intermediate product (IP), and N- and C-
terminal nal products (see Figures S4 and S5 of the
Supporting Information). The identity of these species was
conrmed by ESI-MS, but because of the same mass of the
substrate and the IP, a distinction between them required an
additional comparison of their diering retention times.
We performed the peptide hydrolysis reactions at pH 7.4 and
8.2. The former value was chosen to refer to physiological
conditions; the latter, to easily compare our results with those
Table 4. Parameters of dd Bands of Low-Spin Square-Planar Ni(II) Complexes of Annexin Peptides
UVvis CD
peptide Ni(II) species λmax (nm) ε(dm3×mol1×cm1)λext (nm) Δε(dm3×mol1×cm1)
A11 NiH2L 4N 455 135(1) 449 1.70(4)
519 0.69(1)
NiH3L 4N 454 140(2) 448 1.79(6)
513 0.69(3)
A12 NiL 4N 457 100(1) 391 0.11(3)
443 0.57(3)
497 1.23(8)
559 0.74(5)
4N 457 102(1) 438 0.54(7)
496 1.2(2)
559 0.5(1)
NiH3L 4N 455 100(2) 436 0.60(7)
495 1.4(2)
562 0.6(1)
A13 NiH1L 3N 457 129(7) 442 0.72(4)
501 1.2(3)
NiH2L 4N 432 119(2) 422 1.15(3)
493 1.49(3)
NiH3L 4N 428 120(2) 423 1.11(4)
494 1.56(5)
A21 NiH2L 4N 452 93(3) 433 0.7(1)
493 1.0(2)
568 0.06(3)
NiH3L 4N 448 103(3) 430 1.1(1)
492 1.6(2)
568 0.15(3)
A22 NiH3L 4N 454 121(1) 385 0.05(5)
456 1.6(2)
562 0.07(6)
A81 NiH1L 3N 464 103(5) 448 0.9(1)
506 1.0(1)
569 0.18(5)
NiH2L 4N 448 105(2) 428 0.74(2)
498 1.22(3)
574 0.10(2)
NiH3L 4N 440 104(2) 419 0.84(2)
492 1.32(3)
573 0.08(5)
Since in the result of preliminary calculations we detected that, for A12, two intermediate species, NiH1L and NiH2L, may have very similar
spectra, we merged them in the calculations.
Table 5. Thermodynamic Parameters for Ni(II) Binding to Annexin Peptides Obtained from ITC Experiments in 100 mM
H3BO3,I= 0.1 M, at pH 9.0 and 5 °C
peptide Kaobs
(kJ ×mol1)
(kJ ×mol1)
ΔHNi(II) bind
(kJ ×mol1)
(J ×mol1×K1)
ΔSNi(II) bind
(J ×mol1×K1)
A11 1.1(1) 21.4 21.5 27.6(6) 83.4 176.4 377.1
A12 6.8(8) 131.8 25.7 32.1(3) 87.9 208.1 408.4
A13 1.8(2) 58.9 22.5 39.7(5) 95.5 223.5 424.2
A22 1.6(2) 63.1 22.4 33.7(7) 89.5 202.0 402.3
A81 4.3(3) 100.0 24.7 47.7(5) 103.5 260.2 460.9
The values obtained directly from ITC experiments.
The values calculated on the basis of potentiometric results.
The values calculated with
consideration of the protonation of buer.
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620092002
published earlier by us for other amino acid sequences. The
amount of 4N complexes was low at pH 7.4, as calculated using
potentiometric and spectroscopic results. Under conditions of
hydrolysis experiments, the concentration of 4N complexes did
not exceed 2% of the total peptide concentration. Furthermore,
the product of hydrolysis binds Ni(II) ions much more tightly
than the substrate,
scavenging Ni(II) from the system, so the
concentrations of Ni(II) aqua ions and 4N Ni(II) substrate
complexes decreased systematically during the experiment.
These conditions have a major impact on equations describing
hydrolysis used for the analysis of results of our experiments.
The rate constant equations used previously are valid for higher
pH values, where the degree of complexation is much
In our experimental conditions the hydrolytic 4N
complex is not only consumed by the reaction, but its
concentration is additionally decreased by a shift of complex-
ation equilibrium accompanying the eective dilution of the
reacting system in the course of reaction. In order to
accommodate this eect, we developed new equations (given
in the Supporting Information), which use potentiometry-
derived species distribution data and directly yield the
maximum at the specic temperature, pH-independent rate
constant for the rst step of the reaction (k1Ind), corresponding
to a 100% formation of the hydrolytic 4N complex. The value
corresponding to the rst pH-dependent constant, which we
used and presented previously, can be easily obtained by
multiplying the rst pH independent constant (k1Ind) by the
molar fraction of the hydrolytic 4N complex at the beginning of
the reaction. From such calculation one can obtain the values of
rate constants observed at specic pH, e.g., 7.4 (k17.4) or 8.2
The second step of the reaction, which is the hydrolysis of
the intermediate product described by the k2value, is
independent from the Ni(II) species distribution, although it
is also pH-dependent. The pH dependences of the rst and the
second step of the metal-dependent hydrolysis are dierent.
For the rst step of reaction, it is associated with the formation
of 4N complexes; for the second one, with the concentration of
hydroxide ions, which can catalyze hydrolysis of an ester bond
in the intermediate product.
Thus, in the case of the second
rate constant (k2) we calculated values valid for specic pH.
Because of the low amount of 4N complexes at pH 7.4 (e.g.,
for A11 about 0.16%), we decided to present rate constants
calculated on the basis of experiments performed at pH 8.2
because they provide smaller errors of determination of the
amount of 4N complexes. Rate constants, k1Ind,k18.2, and k28.2,
for the hydrolysis at pH 8.2 are presented in Table 6.
At 37 °C, the highest k1Ind and k18.2 values were for A13,
followed by A81. In the group of peptides with the highest
k28.2 values, we found A81 and A12. The lowest k1Ind,k18.2,
and k28.2 constants were noted for A21 and A11.
We used the linear Arrhenius plot to describe how
temperature inuences the rate constants k18.2 and k28.2 (Figure
5). The values of k18.2 and k28.2 (Figure 5), as well as k1Ind,k17.4,
and k27.4 (Figure S6 of the Supporting Information), fulll the
Arrhenius equation for all studied peptides. Analyzing the
temperature dependence of individual rate constants, we
observe no signicant dierences in slopes between the
peptides. However, the values of k1are more dependent on
temperature than k2.
The hydrolysis of the ester-containing IP is the rate limiting
step of the peptide cleavage for all studied peptides at the
experimental conditions. The amount of IP accumulating in the
initial phase of all hydrolysis experiments is a combined eect
of the rates of the rst and the second step of the reaction.
Specically, the higher the k1in relation to k2, the higher the
amount of IP observed. The highest amount of IP was detected
for A13 (e.g., 85% of the total peptide at pH 8.2 and 37 °C;
Figure S7 of the Supporting Information), as its k18.2 was 17.5
times higher than k28.2. The lowest amount of IP was observed
Figure 4. ITC titration of 2 mM Ac-TKYSKHDMN-am peptide (A1
2), 100 mM H3BO3, 64 mM KNO3, pH 9.0 with 20 mM Ni(NO3)2
and 64 mM KNO3at 5 °C. The upper plot shows raw data from the
experiment; the lower plot shows the absorbed heat (per mol of
injectant) in each injection (black dots), with the tting of the model
with assumed 1:1 interaction stoichiometry (green line).
Table 6. First Order Rate Constants Determined for Ni(II)-Dependent Hydrolysis of the Studied Peptides Calculated on the
Basis of Experiments Performed at pH 8.2
25 °C37°C45°C
peptide k1Ind k18.2 k28.2 k1Ind k18.2 k28.2 k1Ind k18.2 k28.2
A11 0.321(1) 0.081(1) 0.031(1) 1.941(6) 0.492(2) 0.126(1) 6.27(2) 1.589(5) 0.294(1)
A12 1.148(2) 0.648(2) 2.47(2) 6.75(2) 3.811(8) 8.67(4) 19.86(2) 11.22(2) 20.32(4)
A13 21.18(9) 5.910(3) 0.353(1) 100.0(6) 27.9(2) 1.581(6) 480(5) 134(2) 3.86(2)
A21 0.944(2) 0.169(1) 0.145(1) 6.04(2) 1.082(4) 0.455(2) 15.6(1) 2.789(2) 0.968(4)
A22 1.014(5) 0.289(1) 0.547(3) 6.06(2) 1.878(6) 1.770(6) 16.79(5) 4.781(2) 3.467(9)
A81 11.38(5) 3.891(2) 2.523(9) 56.6(2) 19.37(7) 10.33(3) 139.3(7) 47.65(3) 22.08(8)
k1Ind, maximum at specic temperature and pH-independent rate constant for the 1st step of the Ni(II)-dependent hydrolysis; k18.2, the rate
constant for the 1st step of the Ni(II)-dependent hydrolysis at pH 8.2; k28.2, the rate constant for the 2nd step of the Ni(II)-dependent hydrolysis at
pH 8.2.
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620092003
for A12 (e.g., 25% of the total peptide at pH 8.2 and 37 °C),
as its k18.2 was even lower than k28.2.Ask1decreased more
rapidly than k2at pH 7.4, when compared to pH 8.2, the
amounts of observed IP were smaller, ranging from 7% of the
total A12 to 42% of the total A13, at 37 °C. We also
detected a linear temperature dependence of the maximum
amount of IP (except A13 at pH 8.2; Figure S7 of the
Supporting Information), which results from the above-
mentioned dependences of inverse temperature (T1) on the
ln(k1) and ln(k2).
We also determined the half-times of substrate decay (t1/2S)
and the half-times of formation of nal products (t1/2P) (Table
7) because both of these processes could be important from the
toxicological point of view. At pH 7.4 and 37 °Ct1/2S and t1/2P
were the shortest for A13 and A81 and the longest for A2
1 and A11.
Increasing pH to 8.2 does not change the order of peptides
in terms of susceptibility to Ni(II)-dependent hydrolysis.
Nevertheless, we observed a signicant reduction of t1/2S at
pH 8.2 compared to t1/2S at pH 7.4, from about 40 times for
A11 to about 82 times for A22. The t1/2P was shortened
from about 6 times for A11 up to 24 times for A22 peptide.
At pH 8.2, dierences between t1/2S and t1/2P were bigger when
compared to the values observed at pH 7.4.
The hydrolysis of the reference peptide (Ac-GGASRHWKF-
am) was the fastest among all of the studied peptides. At pH
7.4, t1/2S and t1/2P were 2.2 times shorter as compared to
annexin peptides characterized by the shortest t1/2S and t1/2P.At
pH 8.2, these dierences were not so big, and t1/2S of the
reference peptide was similar to t1/2S of A13orA81. The
t1/2P for the reference peptide was comparable to t1/2P for A81
(Table 7).
Characteristics of Complexes. To characterize Ni(II)
complexes, we used methods applied successfully in our
previous research on Ni(II) complexes of peptides susceptible
to Ni(II)-dependent hydrolysis: potentiometry and UVvis
and CD spectroscopies. We supplemented our research
methodology with ITC as an auxiliary method for determi-
nation of binding constants and thermodynamic parameters.
According to our best knowledge, systematic studies of
kinetics of formation of various Ni(II) complexes of His-n(n>
3, where ndenotes position in peptide) and His-3 peptides
were not performed. Nevertheless, it seems clear that His-n
complexes are formed much faster than His-3 complexes. For
example, slow reactivity of the order of minutes to hours is
observed frequently for the latter, often precluding potentio-
metric determination of their stabilities. Such behavior was not
seen in His-ncomplexes studied by us, as well as other research
In the case of annexin peptides we observed the
equilibration of complex formation reactions essentially within
the mixing time (few seconds) of the samples.
The 1N complexes are octahedral species anchored at the
imidazole nitrogen, the only nitrogen donor accessible, due to
its low pKavalue. We detected 1N, 3N, and 4N complexes.
Among 3N complexes we found high-spin species for A21
and A22, and low-spin (square planar) species for A13 and
A81, judged by the absence or presence of a relatively intense
dd band close to 450 nm. The low-spin 3N complexes have
spectra similar to low-spin 4N; however, they are systematically
red-shifted by 24 to 29 nm. It is, according to our best
knowledge, the rst clear-cut account of dd parameters of
low-spin 3N Ni(II) complexes of peptides. We demonstrated
that 3N complexes of annexin model peptides are either low- or
high-spin. In previous studies on Ni(II) complexes of peptides
susceptible to Ni(II)-dependent hydrolysis, also a mixture of
high- and low-spin 3N complexes was observed among Ni(II)
complexes for the same peptide, e.g., for Ac-GASRHAKFL-
The 3N complexes in His peptides are a little mysterious. In
many cases they were found to be inexistent or very minor
species. When observed in amounts sucient to characterize
their spectroscopic properties, they were found to be high-spin,
low-spin, or a mixture of these two cases. In general, the spin
state of a Ni(II) complex depends on the relationship between
the ligand eld strength of the equatorial vs potential axial
ligands (e.g., thermochromism and solvatochromism).
The gradual substitutions of water oxygens with peptide
nitrogens bring the initially octahedral complexes closer to the
spin crossover limit. In complexes involving main chain peptide
nitrogen coordination equatorial 1N and 2N complexes are
always high-spin and 4N complexes are always low-spin. The
Figure 5. Arrhenius plot of the rst rate constants, k18.2 (A), and the
second rate constants, k28.2 (B), describing Ni(II)-dependent peptide
bond hydrolysis at pH 8.2 for peptides: Ac-KALTGHLEE-am (A11),
black squares; Ac-TKYSKHDMN-am (A12), red circles; Ac-
GVGTRHKAL-am (A13),bluetriangles;Ac-KMSTVHEIL-am
(A21), pink triangles; Ac-SALSGHLET-am (A22), green squares;
and Ac-VKSSSHFNP-am (A81), navy triangles.
Table 7. Half-Times of Substrate Decay (t1/2S) and
Formation of Hydrolysis Products (t1/2P) of the Studied
Peptides at 37 °C and pH 7.4 and 8.2
t1/2S (h) t1/2P (h)
peptide pH 7.4 pH 8.2 pH 7.4 pH 8.2
A11 1251 31 1321 231
A12 168 4.1 182 7.7
A1329 0.6 109 14
A21 1076 16 1295 79
A22 610 7.5 702 24
A8145 0.9 60 3.0
13 0.5 28 2.2
The peptide Ac-GGASRHWKF-am, with an amino acid sequence
corresponding to that of the positive nickel hydrolysis control
sequence established in our previous research.
Values for the
fastest processes for annexin peptides in physiological conditions are
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620092004
3N complexes of peptides studied in this work appear to be
near or at the boundary, and so their high-spin (octahedral) or
low-spin (square planar) character is determined by minor
alterations of electronic density of the third nitrogen in the
structure of the complex, that is the amide nitrogen of the
residue preceding the His residue. The diculty of studying
this phenomenon stems from the fact that these 3N complexes
are usually minor species, present only at low concentrations,
due to high cooperativity in the formation of three-ring systems
of nal 4N complexes, as Hill coecient for this process was
always higher than 2 for all studied peptides. In many of the
peptides studied they were not even detected. In order to nd
the rules for the formation of 3N complexes we calculated
protonation-corrected stability constants *K3N according to eq
ββ*= −
og log log
nx n (6)
The βHnLcorresponds to the protonation of the histidine
residue and the βNiHnxLis a Ni(II) cumulative stability constant
of complexes with xnitrogen atoms coordinating metal ion.
The protonation-corrected stability constants enable compar-
isons of complexes having dierent protonation stoichiome-
tries. In particular, they allowed us to compensate for the
numeric eects of deprotonations of nonbonding Tyr and Lys
residues in values of βconstants. In Figure 6 we compared log
*K3N values of high-spin, low-spin, and mixed 3N complexes of
annexin peptides studied here, of hydrolytic peptides used in
our previous research,
and of analogues of the model peptide
of histone H2A.
Generally, among 3N complexes, low-spin
species are the most, and high-spin are the least stable ones.
Complexes with intermediate log *K3N values are a mixture of
high- and low-spin species. The exception is Ac-
GASKHWKFL-am, which forms high-spin 3N complex despite
a high value of log *K3N.
The residue preceding His seems to
be the most important for the 3N complex spin state, and Arg
residue seems to be preferred in this position for low-spin
complexes. Noteworthy, it was demonstrated that Arg side
chain lowers the basicity of its amine nitrogen down to pKa
from a typical value of 7.8. This eect may
also have an inuence on proximate amide nitrogens
coordinating the metal ion. Mlynarz et al. showed that the
lower is the basicity of an amide nitrogen, the higher is the
stability of the Ni(II) complexes.
Altogether, this principle
explains the highest stabilities of 3N complexes with the Arg
While we observed heterogeneity of spectroscopic properties
of 3N complexes, dd band parameters of 4N complexes for all
studied peptides are very similar to each other and consistent
with the results for two species of 4N complex (NiH2L and
NiH3L) of the peptide derived from histone H2A and its
and the peptide derived from the C-terminus of
histone H4.
The protonation-corrected stability constants of the rst (at
the lowest pH) 4N complexes, *K4N, for annexin peptides,
calculated according to eq 6, revealed the highest stability of
Ni(II) complexes with A12, A13, and A81 peptides, with
log *K4N values 27.17, 27.34, and 27.41, respectively. The
remaining log *K4N values are 28.03 for A21, 28.30 for
A22, and 28.46 for A11. The range of log *K4N values
observed for annexin peptides is similar to those obtained in
our previous study on model hydrolytic peptides (from 28.12
to 27.05)
as well as to the Ni(II) complexes of the peptide
derived from histone H2A and its analogues (from 28.58 to
and model peptide of C-terminus of histone H4
Therefore, the 4N Ni(II) complexes containing
imidazole nitrogen and three amide nitrogen donors have
rather uniform stabilities (within 1.6 log units).
When comparing patterns of CD spectra of Ni(II) complexes
of model annexin peptides, we noted that A11 and A13
peptides have the same pattern as observed previously for Ac-
GASRHWKFL-am, a model hydrolytic peptide,
and for
peptide Ac-GTHS-am.
The patterns of CD spectra of A2
1 and A81 peptides are more similar to that observed for Ac-
with an additional small positive band at longer
wavelengths. The A12 peptide has the same CD spectra
pattern as that for Ac-TYTEHA-am, the model peptide from C-
terminus of histone H4,
with more pronounced positive band
at 550 nm. CD spectra of A22 peptide are unusual, as they
generally have only a positive band in the measured spectral
Complex Stabilities and Hydrolysis in the Context of
Biology. The nickel-dependent peptide bond hydrolysis was
observed in viable cultures of CHO (Chinese hamster ovary),
NRK-52 (rat renal tubular epithelium), and HPL1D (human
lung epithelium) cells treated with Ni(II) ions for one to seven
The cleaved bond was found to be in histone H2A
between Glu and Ser residues in sequence ESHHK. It should
be noted that the analytical method used in the cited study
allowed us to observe only the histones. The above-mentioned
value of 28.58 for the protonation-corrected logarithmic
stability constant (log *K4N) for the Ni(II) complex with Ac-
TESHHK-am (a model sequence from histone H2A) was
The comparison of this value with the respective
values for peptides studied here shows that annexin peptides
form more stable Ni(II) complexes, compared to the peptide
derived from H2A. Furthermore, we calculated that the
amounts of 4N hydrolytic complexes for Ac-TESHHK-am
peptide at pH 7.4 and for concentrations used in our hydrolysis
experiments are signicantly (from 5.5 to 65 times, Figure S8 of
the Supporting Information) lower than those observed for
Figure 6. Protonation-corrected logarithmic stability constants of
Ni(II) 3N complexes, log *K3N, and spin states of these complexes.
The considered peptides are annexin peptides marked by green
squares, Ac-GVGTRHKAL-am (TRHK); Ac-KMSTVHEIL-am
(SSHF); and other, previously studied peptides
marked by black
squares, Ac-GASGHAKFL-am (SGHA); Ac-GASAHWKFL-am
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620092005
model peptides of annexins presented here. Since Ni(II) was
shown to bind and cleave H2A in cultured cells, it seems to be
likely that the same processes can also occur for annexin A1,
A2, or A8.
Peptides from histone H2B were thoroughly tested
considering binding of Ni2+ ions.
It was suggested that
formation of such complexes may change conformation of the
whole protein inducing epigenetic and promutagenic eects.
However, taking into account relatively weak binding at
physiological pH, the signicance of these ndings remains to
be established.
Toll-like receptor 4 (TLR4) was proposed by Schmidt et
as a target molecule for Ni2+ ions in nickel allergy. They
stated that a Ni2+ ion bridges two monomers of TLR4 leading
to its direct activation. However, the relevance of that nding in
the context of nickel allergy was called into question in the
recent paper of Vennegaard et al.,
who found that
epicutaneous exposure (resembling common challenge of the
human skin by nickel from, e.g., jewelry) of mice to nickel can
induce allergy independently of TLR4, in contrast to
intradermal injections of nickel performed by Schmidt et al.
This example shows that the mechanisms of nickel allergy are
far from being fully elucidated. There is a need for further
research to reveal the complete spectrum of pathophysiology of
Rates of Hydrolysis. From the physiological point of view,
it is more intuitive to compare the amounts of reaction
products at pH 7.4 and 37 °C, after, e.g., 24 h (Figure 7),
instead of using values of rate constants. In these conditions
more than one-third of the peptide A13 with the sequence
including His293 of annexin A1 is converted to the ester-
containing intermediate product. At the same time, one-fth of
the peptide A81 with the sequence from annexin A8
underwent a complete two-step hydrolysis reaction. In the
case of peptides from annexin A2, the largest amount of IP
(3%) was observed for A22. These results suggest that
annexins A1 and A8, and to some extent also A2, may be
toxicological targets for Ni(II) ions in the context of peptide
bond hydrolysis.
Potential Impact of Ni(II) Binding and Hydrolysis on
Properties of Proteins. The binding of Ni(II) or subsequent
hydrolysis can have several dierent eects; it can (1) change
the conformation of the protein and disturb its native function,
(2) inuence the stability of the protein, and (3) create
epitopes recognized by the immune system as non-native. As
mentioned above, annexins regulate the immune system. Thus,
malfunctioning of the immune system observed, e.g., in allergy
can be induced by any of these three modes of action.
Annexin A1 can inhibit phospholipase A2, cyclooxygenase-2,
and inducible nitric oxide synthase, enzymes involved in
inammatory processes.
Furthermore, one of the potential
Ni(II) binding sites (244SKH246) in annexin A1 is part of
246HDMNKVLDL254, a sequence that was found to inhibit
antigen-induced proliferation of T cells.
Interestingly, the
same site is close to 254LELKGD259 sequence, which is known
as a nuclear export signal.
Finally, all three potential Ni(II)
binding sites are in the proximity of Ca2+ binding residues.
Thus, one can assume that binding of Ni(II) and subsequent
change of the conformation, being a result either of a binding or
an acyl shift, as well as hydrolysis of peptide bond in annexin
A1 may have an inuence on important physiological processes.
The N-terminal part of annexin A2, containing potential
Ni(II) binding residues 3TVH5, consists of a nuclear export
sequence: 4VHEILCKLSL13.
Furthermore, residues Val4, Ile7,
Leu8, and Leu11 participate in the interaction with the
S100A10 protein.
There is a redox active Cys9 residue in
the same region. This cysteine residue is reversibly oxidized by
H2O2and reduced by the thioredoxin system; thus, annexin A2
is a probable antioxidant molecule.
Annexin A2 accumulates
in the nucleus in response to oxidative stress, and this is
probably one of the means of DNA protection. In the absence
of oxidative stress, annexin A2 is exported from the nucleus.
In this context, it is important to mention that Ni(II)
complexes with peptides were found to induce DNA breakage
in the presence of H2O2.
Thus, binding of Ni(II) to
3TVH5may have an inuence on these processes. The Ni(II)-
dependent cleavage of peptide bond between Ser2 and Thr3
could also transform isoform 2 of annexin A2 with the longer
N-terminal part to the analogue of isoform 1. As mentioned
above, another potential Ni(II) binding site, 92SGH94, is close
to Ca2+ binding residues. In the result, Ni(II) binding may also
impair calcium-dependent functions of annexin A2.
The binding of Ni(II) and peptide bond hydrolysis in
annexin A8 is probably most relevant for smokers and other
humans exposed to high nickel level in the air, as the highest
levels of this protein was observed in human lung
The results presented above permit us to consider the
toxicological impact of three chemical processes diering in
their velocity: (1) Ni(II) binding, (2) formation of the
intermediate product, and (3) formation of the nal products.
Chelation of nickel by amino acid sequences including the His
residue in position further than 3 is fast (in the range of
seconds, as mentioned above), and its duration is correlated
with a short contact of human skin with, e.g., coins. It has to be
emphasized that even such short contact is sucient to elicit
allergic reaction.
However, formation of IP and nal products
is slower. It seems to correlate with the fact that the nickel
allergy is usually linked with type IV hypersensitivity, which is
known to develop days after the contact with allergen.
Perspective. We demonstrated that all peptides from
human annexins studied here undergo Ni(II)-dependent
hydrolysis. Analysis of 3D structures of annexins A1, A2, and
A8 showed that potential hydrolytic sites are accessible for
Ni(II) ions and can adopt conformations required for the
square planar complex formation in native proteins. The next
Figure 7. Molar fraction of an intermediate product (blue) and
hydrolysis nal products (red) after 24 h at pH 7.4 and 37 °C, for
peptides Ac-KALTGHLEE-am (A11), Ac-TKYSKHDMN-am (A1
2), Ac-GVGTRHKAL-am (A13), Ac-KMSTVHEIL-am (A21), Ac-
SALSGHLET-am (A22), and Ac-VKSSSHFNP-am (A81).
Chemical Research in Toxicology Article |Chem. Res. Toxicol. 2014, 27, 199620092006
step of our research is to react whole proteins with Ni(II) ions,
followed by experiments on cell lines. These studies will allow
us to verify possible toxicological eects of Ni(II)-dependent
hydrolysis on the structure and functions of annexins.
SSupporting Information
Detailed description of kinetics of the metal-dependent
hydrolysis; species distributions of Ni(II) complexes for
considered binding models, including rmsd results; deconvo-
luted UVvis and CD spectra of low-spin Ni(II) complexes;
representative HPLC chromatograms and the time dependence
of relative amount of peptide species during hydrolysis;
Arrhenius plots for hydrolysis performed at pH 7.4; a
temperature dependence of the maximum amount of IP; a
comparison of 4N complexes amounts at pH 7.4. This material
is available free of charge via the Internet at
Corresponding Author
*Tel: +48-22-592-5766. Fax: +48-22-659-4636. E-mail:
This study was partially nanced by Polish National Science
Centre, Grant No. DEC-2011/01/D/NZ1/03501. This work
was also supported in part by the project Metal dependent
peptide hydrolysis. Tools and mechanisms for biotechnology,
toxicology and supramolecular chemistry, carried out as part of
the Foundation for Polish Science TEAM/2009-4/1 program,
conanced from European Regional Development Fund
resources within the framework of Operational Program
Innovative Economy. The equipment used was sponsored in
part by the Centre for Preclinical Research and Technology
(CePT), a project cosponsored by European Regional
Development Fund and Innovative Economy, The National
Cohesion Strategy of Poland.
The authors declare no competing nancial interest.
DCM, dichloromethane; DIEA, N,N-diisopropylethylamine;
HBTU, O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium
hexauorophosphate; ITC, isothermal titration calorimetry;
IP, intermediate product; P, products; S, substrate; TIS,
triisopropylsilane; TFA, triuoroacetic acid; TLR4, Toll-like
receptor 4; Xaa, any amino acid residue except proline; Yaa,
Zaa, any amino acid residue
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... The Ser/Thr-Xaa-His sequence occurs in several human proteins. We have shown that histone H2A [18], annexins A1, A2, and A8 [19], alpha-1-antitrypsin [20], phospholipid scramblase 1, Sam68-like mammalian protein 2, and CLK3 kinase [21] contain surface-exposed fragments cleavable by Ni(II) ions. Ni(II)-prone sequences are also present in all of the melatonin biosynthesis pathway enzymes, i.e., tryptophan 5-hydroxylase 1 (TPH1), aromatic-L-amino-acid decarboxylase (AADC), serotonin N-acetyltransferase (SNAT), and acetylserotonin O-methyltransferase (ASMT). ...
... The Ser/Thr-Xaa-His sequence occurs in several human proteins. We have shown that histone H2A [18], annexins A1, A2, and A8 [19], alpha-1-antitrypsin [20], phospholipid scramblase 1, Sam68-like mammalian protein 2, and CLK3 kinase [21] contain surfaceexposed fragments cleavable by Ni(II) ions. Ni(II)-prone sequences are also present in all of the melatonin biosynthesis pathway enzymes, i.e., tryptophan 5-hydroxylase 1 (TPH1), aromatic-L-amino-acid decarboxylase (AADC), serotonin N-acetyltransferase (SNAT), and acetylserotonin O-methyltransferase (ASMT). ...
... Comparing stability constants among different peptides (having different protonation stoichiometries) is possible after the correction for the protonation of the ligand, according to the Equation (1) [19]: ...
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Nickel is toxic to humans. Its compounds are carcinogenic. Furthermore, nickel allergy is a severe health problem that affects approximately 10–20% of humans. The mechanism by which these conditions develop remains unclear, but it may involve the cleavage of specific proteins by nickel ions. Ni(II) ions cleave the peptide bond preceding the Ser/Thr-Xaa-His sequence. Such sequences are present in all four enzymes of the melatonin biosynthesis pathway, i.e., tryptophan 5-hydroxylase 1, aromatic-l-amino-acid decarboxylase, serotonin N-acetyltransferase, and acetylserotonin O-methyltransferase. Moreover, fragments prone to Ni(II) are exposed on surfaces of these proteins. Our results indicate that all four studied fragments undergo cleavage within tens of hours at pH 8.2 and 37 °C, corresponding with the conditions in the mitochondrial matrix. Since melatonin, a potent antioxidant and anti-inflammatory agent, is synthesized within the mitochondria of virtually all human cells, depleting its supply may be detrimental, e.g., by raising the oxidative stress level. Intriguingly, Ni(II) ions have been shown to mimic hypoxia through the stabilization of HIF-1α protein, but melatonin prevents the action of HIF-1α. Considering all this, the enzymes of the melatonin biosynthesis pathway seem to be a toxicological target for Ni(II) ions.
... The involvement of imidazole nitrogens is confirmed by characteristic charge transfer transitions detected in CD spectra: N im →Ni(II), at about 260 nm. The resulting complexes with a 4N donor set have the maximum absorbance (λmax) of d-d bands at 410 nm and 478 nm for Hpnl1 peptide and 412 nm and 478 nm for Hpnl2 peptide [15,28]. There is no shift to the shorter or longer wavelengths with the addition of nickel ions up to two equivalents of Ni(II), which reveals that no additional metal ion is bound at this pH (7.4). ...
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Combined potentiometric titration and isothermal titration calorimetry (ITC) methods were used to study the interactions of nickel(II) ions with the N-terminal fragments and histidine-rich fragments of Hpn-like protein from two Helicobacter pylori strains (11637 and 26695). The ITC measurements were performed at various temperatures and buffers in order to extract proton-independent reaction enthalpies of nickel binding to each of the studied protein fragments. We bring up the problem of ITC results of nickel binding to the Hpn-like protein being not always compatible with those from potentiometry and MS regarding the stoichiometry and affinity. The roles of the ATCUN motif and multiple His and Gln residues in Ni(II) binding are discussed. The results provided the possibility to compare the Ni(II) binding properties between N-terminal and histidine-rich part of Hpn-like protein and between N-terminal parts of two Hpn-like strains, which differ mainly in the number of glutamine residues.
... The Annexins common functions are the Ca +2 -dependent phospholipid binding, and the inhibition of phospholipase A 2 and prothrombinase activity [53]. It is demonstrated that Ni +2 can bind to this protein by a sequence of amino acids ( 18 Ser-Ser-His 20 ) while Hg binding could be possible in the same sequence [54]. As we said before, Hg has the possibility to bind His [51]. ...
The interest in inorganic Hg toxicity and carcinogenicity has been pointed to target organs such as kidney, brain or placenta, but only a few studies have focused on the mammary gland. In this work, analytical combination techniques (SDS-PAGE followed by CV-AFS, and nanoUPLC-ESI-MS/MS) were used to determine proteins that could bind Hg in three human mammary cell lines. Two of them were tumorigenic (MCF-7 and MDA-MB-231) and the other one was the non-tumorigenic cell line (MCF-10A). There are no studies that provide this kind of information in breast cell lines with IHg treatment. Previously, we described the viability, uptake and the subcellular distribution of Hg in human breast cells and analysis of RNA-seq about the genes that encode proteins which are related to cytotoxicity of Hg. This work provides important protein candidates for further studies of Hg toxicity in the mammary gland, thus expanding our understanding of how environmental contaminants might affect tumor progression and contribute with future therapeutic methods.
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Deficiency in a principal epidermal barrier protein, filaggrin (FLG), is associated with multiple allergic manifestations, including atopic dermatitis and contact allergy to nickel. Toxicity caused by dermal and respiratory exposures of the general population to nickel-containing objects and particles is a deleterious side effect of modern technologies. Its molecular mechanism may include the peptide bond hydrolysis in X 1 -S/T-c/p-H-c-X 2 motifs by released Ni ²⁺ ions. The goal of the study was to analyse the distribution of such cleavable motifs in the human proteome and examine FLG vulnerability of nickel hydrolysis. We performed a general bioinformatic study followed by biochemical and biological analysis of a single case, the FLG protein. FLG model peptides, the recombinant monomer domain human keratinocytes in vitro and human epidermis ex vivo were used. We also investigated if the products of filaggrin Ni ²⁺ -hydrolysis affect the activation profile of Langerhans cells. We found X 1 -S/T-c/p-H-c-X 2 motifs in 40% of human proteins, with the highest abundance in those involved in the epidermal barrier function, including FLG. We confirmed the hydrolytic vulnerability and pH-dependent Ni ²⁺ -assisted cleavage of FLG-derived peptides and FLG monomer, using in vitro cell culture and ex-vivo epidermal sheets; the hydrolysis contributed to the pronounced reduction in FLG in all of the models studied. We also postulated that Ni-hydrolysis might dysregulate important immune responses. Ni ²⁺ -assisted cleavage of barrier proteins, including FLG, may contribute to clinical disease associated with nickel exposure.
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NiO nanoparticles and non-stoichiometric black NiO were shown to be effective sources of Ni ²⁺ ions causing sequence-selective peptide bond hydrolysis. NiO nanoparticles were as effective in this reaction as their...
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Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr−Xaa−His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68‐like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.
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Three extensively used metals, cadmium, chromium and nickel, are established human carcinogens. The elucidation of the molecular and cellular mechanisms underlying the carcinogenicity of these metals has centered mostly on the signalling pathways that regulate cellular growth, differentiation and death. Unfortunately, our understanding of the involvement of these pathways in metal-induced carcinogenesis is still very incomplete. More recently, research has extended to include the impact of these metals on mechanisms not traditionally associated with cancer, but that are now increasingly viewed as playing a critical role in carcinogenesis. Among them is the stress response, a highly conserved mechanism employed by all cells for protection against protein damage. Indeed, all three metals induce proteotoxic stress, which warrants following this line of research. The present chapter will critically review published studies on the impact of carcinogenic metals on the expression of the heat shock protein 90 family (HSP90), one of the protein families that mediate the stress response. HSP90 has been consistently found to be overexpressed in many types of cancer and, significantly, HSP90 overexpression has been correlated with increased tumor growth, metastatic potential and resistance to chemotherapy.
The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3′-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.
Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 Cu(II) is bound to the model peptide hCtr1_1-14 via an ATCUN motif and such complexes are strong enough to collect Cu(II) from albumin, supporting the potential physiological role of Cu(II) binding to hCtr1.
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In this work we demonstrate that the previously described reaction of sequence specific Ni(II)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys2His2 zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(II) ions. It results in loss of the Zn(II) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(II)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(II)-susceptible sequence between the edited zinc finger and an affinity tag allows for easy removal of the latter sequence by Ni(II) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(II) ions. Mass spectra revealed that a Ni(II) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(II)-dependent protein hydrolysis is influenced by the Ni(II) concentration, pH and temperature of the reaction provides a platform for novel regulated DNA effector design.
The conformational preferences and the solution structure of AnXII N 3 1 , a peptide corresponding to the full-length sequence (residues 1-31) of the human annexin II N-terminal tail domain, were investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AnxII N 3 1 adopts a mainly α-helical conformation in hydrophobic or membrane-mimetic environments, while a predominantly random structure is adopted in aqueous buffer. In contrast to previous results of the annexin I N-terminal domain peptide [Yoon et al. (2000) FEBS Lett. 484, 241-245], calcium ions showed no effect on the structure of AnxII N 3 1 . The NMR-derived structure of AnXII N 3 1 in 50% TFE/water mixture showed a horseshoe-like fold comprising the N-terminal amphipathic α-helix, the following loop, and the C-terminal helical region. Together, the results establish the first detailed structural data on the N-terminal tail domain of annexin II, and suggest the possibility of the domain to undergo Ca 2 + -independent membrane-binding.
Epidemiological evidence suggests that certain paternal exposures to metals may increase the risk of cancer in the progeny. This effect may be associated with promutagenic damage to the sperm DNA. The latter is packed with protamines which might sequester carcinogenic metals and moderate the damage. Human protamine P2 has an amino acid motif at its N-terminus that can serve as a heavy metal trap, especially for Ni(II) and Cu(II). We have synthesized a pentadecapeptide modeling this motif, Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-CysSer-Arg-amide (HP21‐15) and described its complexes with Ni(II) and Cu(II), including their capacity to mediate oxidative DNA degradation [Bal et al. (1997) Chem. Res. Toxicol., 10, 906‐914 and 915‐921]. In the present study, effects of HP21‐15 on Ni(II)- and Cu(II)-mediated DNA oxidation by H2O2 at pH 7.4 were investigated in more detail using the circular plasmid pUC19 DNA as a target, and the single/double-strand breaks and production of oxidized DNA bases, as end points. Ni(II) alone was found to promote oxidative DNA strand scission (mostly single strand breaks) and base damage, while Cu(II) alone produced the same effects, but to a much greater extent. Both metals were relatively more damaging to the pyrimidine bases than to purine bases. HP21‐15 tended to increase the Ni(II)/H2O2-induced DNA breakage. In sharp contrast, the destruction of DNA strands by Cu(II)/H2O2 was almost completely prevented by HP21‐15. The effect of HP21‐15 on the oxidative DNA base damage varied from a limited enhancement (5-hydroxyhydantoin and thymine glycol) to slight suppression (5-hydroxycytosine, 5-hydroxyuracil, 8-oxoguanine, 8-oxoadenine, 2-hydroxyadenine, fapyguanine and fapyadenine) toward Ni(II)/H2O2. HP21‐15 strongly suppressed the oxidative activity of Cu(II)/H 2O2 in regard to all bases in DNA. Consistently with the Abbreviations: 2-OH-Ade, 2-hydroxyadenine (isoguanine); 5-OH-Cyt, 5-hydroxycytosine; 5-OH-Hyd, 5-hydroxyhydantoin; 8-oxo-Ade, 7,8-dihydro8-oxoadenine; 8-oxo-dG, 7,8-dihydro-8-oxo-29-deoxyguanosine (8-oxo-29deoxyguanosine); 8-oxo-Gua, 7,8-dihydro-8-oxoguanine (8-oxoguanine); BSTFA, bis(trimethylsilyl)trifluoroacetamide; dG, 2 9-deoxyguanosine; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ESR, electron spin resonance; HP2, human protamine 2; HP21‐15, Arg-Thr-His-GlyGln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide; PBS, phosphatebuffered saline; TBE buffer, 0.1 M Tris, 0.09 M boric acid and 0.001 M EDTA, pH 8.4; ThyGlycol, thymine glycol.
Background Nickel allergy is common worldwide. It is associated with hand dermatitis, and sensitization is often induced by nickel-releasing jewellery. The European Union (EU) introduced legislation to control nickel content and release from jewellery and other consumer items through the EU Nickel Directive 1994, which came into force in 2001 and is now part of the REACH regulation. Objectives To examine the effects of the EU nickel regulations on the prevalence of nickel allergy in four European countries. Methods Nickel patch-test data from 180 390 patients were collected from national databases in Denmark, Germany, Italy and the U.K. from between 1985 and 2002 to 2010. Patients with suspected allergic contact dermatitis who had been patch tested with nickel sulfate 5% in petrolatum were included in the analysis. The main outcomes studied were the percentage of positive results to nickel patch tests, and changes in trends with time in an age- and sex-stratified analysis. ResultsA statistically significant decrease in nickel allergy was observed in Danish, German and Italian women aged below 30 years. In female patients in the U.K. this was observed between 2004 and 2010. In young men, a statistically significant decrease in nickel allergy was observed in Germany and the U.K., whereas a nonsignificant increase was observed in Italy. Conclusions There has been a reduction in the prevalence of nickel allergy in young women, contemporaneous with the introduction of the nickel regulation. A reduction is also suggested in men in Germany and the U.K. A causative effect of the regulatory intervention is the most likely explanation.
Nickel is widely applied in industrial settings and Ni(II) compounds have been classified as group one human carcinogens. The molecular basis of Ni(II) carcinogenicity has proved complex, for many stress response pathways are activated and yield unexpected Ni(II)-specific toxicology profile. Ni(II)-induced toxicogenomic change has been associated with altered activity of HIF, p53, c-MYC, NFκB and iron and 2-oxoglutarate-dependent dioxygenases. Advancing high-throughput technology has indicated the toxicogenome of Ni(II) involves crosstalk between HIF, p53, c-MYC, NFκB and dioxygenases. This paper is intended to review the network of Ni(II)-induced common transcription-factor-governed pathways by discussing transcriptome alteration, its governing transcription factors and the underlying mechanism. Finally, we propose a putative target network of Ni(II) as a human carcinogen.
Background Several attempts to establish a model in mice that reflects nickel allergy in humans have been made. Most models use intradermal injection of nickel in combination with adjuvant to induce nickel allergy. However, such models poorly reflect induction of nickel allergy following long-lasting epicutaneous exposure to nickel.Objective To develop a mouse model reflecting nickel allergy in humans induced by epicutaneous exposure to nickel, and to investigate the mechanisms involved in such allergic responses.Methods Mice were exposed to NiCl2 on the dorsal side of the ears. Inflammation was evaluated by the swelling and cell infiltration of the ears. T cell responses were determined as numbers of CD4+ and CD8+ T cells in the draining lymph nodes. Localization of nickel was examined by dimethylglyoxime staining.ResultsEpicutaneous exposure to nickel results in prolonged localization of nickel in the epidermis, and induces nickel allergy in mice. The allergic response to nickel following epicutaneous exposure is MyD88-dependent and interleukin (IL)-1 receptor-dependent, but independent of toll-like receptor (TLR)-4.Conclusion This new model for nickel allergy that reflects epicutaneous exposure to nickel in humans shows that nickel allergy is dependent on MyD88 and IL-1 receptor signalling, but independent of TLR4.
Copper(II) and nickel(II) complexes of an octapeptide (Ac-MKHMGTHS-NH2) and its N-terminally lengthened derivatives (Ac-GGMKHMGTHS-NH2 and Ac-GDMKHMGTHS-NH2) have been studied by potentiometric and spectroscopic measurements. The amino acid sequences of the peptides correspond to the metal binding domains of H-96 and H-111 residues in the native prion fragments (GT(96)HS and (MKHM)-H-111) but these domains are in the opposite order in the model peptides. The results revealed that both histidyl moieties can be effective metal binding sites. Evaluation of CD spectra made it possible to assess the ratio of coordination isomers of mononuclear complexes. The preference both for copper(II) and nickel(II) binding at the GTHS site was obtained in all cases. In the case of nickel(II) this is in a good agreement with the previous results reported for the prion fragments but the opposite distribution was obtained for copper(II). These data strongly support that nickel(II) prefers the binding to the specific sites of peptides best suited for square planar coordination, while the overall stability of copper(II) peptide complexes is finely tuned by the secondary effects of distant side chains. Moreover, the formation of heteropolynuclear copper(II)-nickel(II)-peptide complexes has also been detected and it was found that the addition of nickel(II) to the mononuclear copper(II) species can redistribute the coordination of copper(II) among the available metal binding sites. (C) 2014 Published by Elsevier Ltd.
Potentiometry and UV-vis and circular dichroism spectroscopies were applied to characterize Cu(II) coordination to the Ac-GASRHWKFL-NH2 peptide. Using HPLC and ESI-MS, we demonstrated that Cu(II) ions cause selective hydrolysis of the Ala-Ser peptide bond in this peptide and characterized the pH and temperature dependence of the reaction. We found that Cu(II)-dependent hydrolysis occurs solely in 4N complexes, in which the equatorial coordination positions of the Cu(II) ion are saturated by peptide donor atoms, namely, the pyridine-like nitrogen of the His imidazole ring and three preceding peptide bond nitrogens. Analysis of the reaction products led to the conclusion that Cu(II)-dependent hydrolysis proceeds according to the mechanism demonstrated previously for Ni(II) ions ( Kopera , E. ; Krężel , A. ; Protas , A. M. ; Belczyk , A. ; Bonna , A. ; Wysłouch-Cieszyńska , A. ; Poznański , J. ; Bal , W. Inorg. Chem. 2010 , 49 , 6636 - 6645 ). However, the pseudo-first-order reaction rate found for Cu(II) is, on average, 100 times lower than that for Ni(II) ions. The greater ability of Cu(II) ions to form 4N complexes at lower pH partially compensates for this difference in rates, resulting in similar hydrolytic activities for the two ions around pH 7.
Background The possible impact of metal release from coronary artery stents has, with their increased use, become a concern.Objectives To study in vitro metal release in biologically relevant milieu from coronary stents made of different alloys.Materials and methodCoronary stents in common use in a department of cardiology at the time of the study were tested. A previously described in vitro technique was used, whereby the stents were kept in the extraction media for a week. Two different extraction media were used to show the necessity of studying the actual biological surrounding of the implant when metal release is investigated. Metal release was determined with atomic absorption spectrometry.ResultsIn this study, we show metal release from stents after immersion in extraction media of artificial sweat and cysteine solution, as illustrative media.Conclusion Metal release from coronary stents is shown. The magnitude of release is influenced by several factors. The extent to which metal release in vitro has potential biological effects, in terms of elicitation of an allergic reaction or induction of sensitization, in vivo needs to be explored. However, as metal release from an implant in a biologically appropriate medium has been established, better risk assessments in relation to delayed hypersensitivity may be undertaken.
In our previous research we demonstrated the sequence specific peptide bond hydrolysis of the R1-(Ser/Thr)-Xaa-His-Zaa-R2 in the presence of Ni(II) ions. The molecular mechanism of this reaction includes an N-O acyl shift of the R1 group from the Ser/Thr amine to the side chain hydroxyl group of this amino acid. The proposed role of the Ni(II) ion is to establish favorable geometry of the reacting groups. In this work we aimed to find out whether the crucial step of this reaction--the formation of the intermediate ester--is reversible. For this purpose we synthesized the test peptide Ac-QAASSHEQA-am, isolated and purified its intermediate ester under acidic conditions, and reacted it, alone, or in the presence of Ni(II) or Cu(II) ions at pH 8.2. We found that in the absence of either metal ion the ester was quickly and quantitatively (irreversibly) rearranged to the original peptide. Such reaction was prevented by either metal ion. Using Cu(II) ions as CD spectroscopic probe we showed that the metal binding structures of the ester and the final amine are practically identical. Molecular calculations of Ni(II) complexes indicated the presence of steric strain in the substrate, distorting the complex structure from planarity, and the absence of steric strain in the reaction products. These results demonstrated the dual catalytic role of the Ni(II) ion in this mechanism. Ni(II) facilitates the acyl shift by setting the peptide geometry, and prevents the reversal of the acyl shift, by stabilizing subsequent reaction products.